(1) Field of the Invention
The invention relates to the production of heterologous proteins in gram-positive bacteria, in particular lactic acid bacteria.
(2) Description of the Background
Besides their conventional uses in the agrofoods industry, lactic acid bacteria are currently increasingly used as host cells for the production of heterologous proteins of interest. These proteins of interest can be very varied in nature, and it is therefore desirable to have as large a choice as possible of expression tools in order to be able to optimize the production thereof as a function of the specificities of each one of them.
In general, it is necessary to use strong promoters that make it possible to obtain a sufficient level of expression of the gene of interest. In certain cases, constitutive promoters can be used; in other cases (for example when the product of the gene of interest is toxic for the host bacterium or there is a risk of it interfering with the metabolism thereof), it is preferable to use inducible promoters that make it possible to initiate or to stop the expression at the desired time.
Although lactic acid bacteria have many genes whose transcription is regulated by various factors, at the current time, there is only a relatively restricted choice of inducible promoters that can be used in practice for constructing expression cassettes for genes of interest (for review, cf. D E VOS, Curr. Op. Microbial., 2, 289-295, 1999). In fact, this use requires not only that the promoters concerned can be regulated, but also that there exists a sufficient expression differential between the various induction states; ideally, the expression should reach a high level under induction conditions and should be able to be completely blocked under non-induction conditions. In addition, it is necessary to be able to readily control the factors involved in the regulation of these promoters.
In previous studies, aimed at identifying exported proteins from L. lactis (POQUET et al., J. Bacteriol., 180, 1904-1912, 1998), the inventors cloned, by fusion with the reporter gene ΔSPNuc, a fragment of genomic DNA of the L. lactis strain MG1363, comprising a gene called, at the time, nlp3 (New LipoProtein 3), the product of which exhibits homologies with an S. pneumoniae protein involved in the transport of metals. The sequence of this fragment is available on GENBANK under the number U95834.
The inventors also observed that the nlp3 gene was negatively regulated by divalent metal cations, in particular Zn2+ (POQUET et al. “Use of a new reporter tool to demonstrate metal regulation of nlp3, a gene putatively involved in metal uptake in Lactococcus lactis”; 6th Symposium on Lactic Acid Bacteria, Veldhoven, The Netherlands, Sep. 19-23, 1999).
Furthermore, in the context of the complete sequencing of the L. lactis IL1403 genome, the nlp3 gene, renamed zitS, was identified as a constituent of an operon, called zitRSQP (BOLOTIN et al., Antonie van Leeuwenhoek, 76, 27-76, 1999; BOLOTIN et al., Genome Res. 11, 731, 2001). By homology with known sequences, putative functions in zinc transport were attributed to the genes of this operon. Thus, the product of the zitP gene is thought to constitute the permease of the transport system, the products of the zitS gene and of the zitQ gene are thought to ensure, respectively, binding with the substrate and binding with ATP, and the product of the zitR gene, which exhibits homologies with the marR transcriptional repressor family, is thought to be involved in the regulation of zinc transport.
Up until this point, use of the zitRSQP operon regulatory system for controlling the expression of heterologous genes had not been envisioned. In fact, although a negative regulation may be initiated by the addition of zinc (POQUET et al., 1999, mentioned above), the basal level of expression observed in the absence of this negative regulation did not appear to be sufficient to allow satisfactory production of proteins of interest. In addition, it was not known whether the putative repressor ZitR was effectively involved in repression of the expression of this operon, or whether other regulators, in particular the pleiotropic flp regulators, described as being involved in the regulation of zinc transport in L. lactis (GOSTICK et al., Mol. Microbiol., 31, 1523-35, 1999; SCOTT et al., FEMS Microbiol. Lett., 192, 85-89, 2000), could also be involved, either as corepressors or, conversely, as possible activators.